Fig 1.
Cardiospheres contain pluripotent mesenchymal cells.
a, Representative fluorescence microscopy image of intact cardiosphere (C-sphere) spontaneously generated from the RV explant from a patient with tetralogy of Fallot (TF) following 4 weeks in tissue culture. Scale bar, 300 μm. Inset, x30 magnified confocal image of c-kit-positive cells (in red) within the shell of the C-sphere. Scale bar, 10 μm. b, Representative confocal micrograph of cryosectioned C-sphere obtained from the RV heart sample of patient with TF immunostained for vimentin (in green) merged with c-kit (in red). Scale bar, 20 μm. c, Representative images of C-sphere-derived cells immunostained with various differentiation markers, including early cardiogenic marker: MEF-2C, stem cell marker c-kit, the endothelial lineage marker von Willebrands factor (vWF), mesenchymal marker vimentin, fibroblast marker fibroblast-specific protein 1 (Fsp-1), and smooth muscle cell myosin (SMM) marker. Scale bar, 100 μm. Nuclei were marked with DAPI staining (in blue).
Fig 2.
The RV from HLHS exhibits depleted cellular content.
a, Bright-field images of spontaneously generated C-spheres obtained from HLHS patient (7 month of age) and patient with dilated cardiomyopathy (DCM) (6 months of age). Scale bar, 200 μm. b, Bright-field images of C-spheres obtained from both DCM-RV and DCM-LV heart samples propagated in multiple passages. Scale bar, 400 μm. c, Representative fluorescence microscopy images of C-spheres derived from the LV and the RV of HLHS patient immunostained for vimentin (in green) and c-kit (in red). Scale bar 100 μm. d, Representative fluorescence microscopy images of C-sphere-derived cells (CDCs) obtained from the RV and the LV of end-stage HLHS immunostained for various differentiation markers, including stem cell antigen c-kit, fibronectin, fibroblast marker Fsp-1, early cardiogenic marker (β-myosin heavy chain, MF-20) and mesenchymal cell marker vimentin. Scale bar, 100 μm. Nuclei were marked with DAPI staining (in blue). e, Percentage of c-kit and vimentin positive cells in CDCs isolated from HLHS-LV versus CDCs isolated from HLHS-RV. Bar graphs represent mean values ± SEM, n = 10 random fields. c-kit, ** P value <0.0018; vimentin, **** P value<0.0001.
Table 1.
Ventricular C-sphere content.
Fig 3.
The RV in HLHS becomes deficient in cardiovascular lineage progenitor cells during the evolution of heart failure.
a, Representative fluorescence microscopy images of cryosectioned HLHS samples showing the cellular content in the RV and the LV at transplantation, immunostained for cardiac markers α-actinin and β-myosin heavy chain (MF20), progenitor cell marker c-kit, the early cardiogenic marker MEF-2C and the endothelial cell marker CD31. Scale bar, 150 μm. b, Percentage of c-kit-, MEF-2C- and CD31-positive cells in cryopreserved tissue samples isolated from HLHS-LV versus the HLHS-RV. Bar graphs represent mean values ± SEM, n = 10 random fields. c-kit * P value<0.0001; MEF-2C * P value<0.029; CD31 P value = NS. c, Representative confocal micrographs of cryosectioned tissues obtained from the RV and the LV of HLHS patient immunostained for fibronectin (in red) and α-actinin (in green). d, Representative confocal micrographs of cryosectioned tissues obtained from the RV and the LV of HLHS patient immunostained for vimentin (in red) and α-actinin (in green). Image insets represent 3 X magnification. Scale bar, 20 μm. Bar graph, percentage of α-actinin- and vimentin-double positive cells in cryopreserved tissue samples isolated from HLHS-LV versus HLHS-RV. Data are mean values ± SEM, n = 10 random fields. ** P value<0.0057. e, Western Blot analysis of protein expression levels of cardiac lineage markers Nkx.2.5 and MEF-2C, and that of integrin-linked kinase (ILK) in the LV compared to the RV in HLHS, and in RV and LV in DCM (age 6 months). f, Representative fluorescence microscopy images of cryosectioned RV cardiac tissue isolated from neonatal patient (age, day 8) immunostained with MF-20, vWF, c-kit, vimentin and CD31 markers. The neonatal RV in HLHS resembled the LV in HLHS at transplantation, based on the abundant presence of c-kit, vimentin, and the endothelial markers (vWF; CD31). Scale bar, 80 μm. Nuclei were labeled with DAPI staining (in blue).
Table 2.
Correlation between echocardiographic ventricular function and stem cell content.
Fig 4.
DNA replication occurs in stromal cells but not in existing cardiomyocytes in HLHS and in TF.
a, ventricular samples from HLHS RV, HLHS LV, and TF immunostained for cardiomyocyte α-actinin and PH3. b, the % of non-cardiomyocytes that were PH3-positive was higher in TF than in HLHS. PH3+ nuclei with minimum 90% cardiomyocyte overlap, considered as PH3+ cardiomyocytes (CM), were not detectable in any heart samples. Counts in non-CMs were normalized to total nuclei per field of view, shown as mean + SEM, and compared by one-way ANOVA and post-hoc Tukey's pairwise comparisons test. Scale bar = 20 microns. Blue = DAPI, red = PH3, green = α-actinin.
Fig 5.
Schematic representation of differentiation capacity in LV and RV in HLHS.
a, C-spheres generated from LV in HLHS are comprised of continuously layered double-positive c-kit+ and vimentin+ cells. C-spheres generate cardiosphere-derived cells which possess adherence properties consistent with a cardiac mesenchymal cell (CMC), and exhibit differentiation capacity into multiple cardiovascular lineage cell types as shown. Immature cardiomyocytes are more frequent in the LV than the RV. b, Excessive RV mechanical stress results in depletion of regenerative capacity in the RV in HLHS. CMCs within HLHS-RV tissue are sparsely distributed on the surface of the tissue fragments and create a non-spherical shape. These cells are vimentin-positive and c-kit-negative and become incompetent for differentiation as a result of stress-induced senescence. CMCs, cardiac mesenchymal cells; EC, endothelial cells; CM, cardiomyocyte; FB, fibroblast; SMC, smooth muscle cell.